3-dimensionial (3d) tissue-engineered muscle for tissue restoration

ABSTRACT

The present disclosure provides solid collagen constructs and tissue compositions, wherein a polymerizable collagen solution or suspension is extruded in the presence or absence of cells to formed an aligned architecture comprising solid collagen constructs such as those made with fibrillar collagen. Methods of using and of manufacturing solid collagen constructs and tissue compositions, where the component collagen is solid fibrillar collagen and cells are preferentially aligned, are also provided.

GOVERNMENT RIGHTS

This invention was made with government support under DC014070 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF INVENTION

This disclosure relates to tissue restoration applications includingcompositions for such applications and methods for making suchcompositions. This disclosure includes tissue-engineered solid collagenconstructs that may be made with, or without, cells used for tissuerestoration as well as an in-vitro testing platform.

BACKGROUND

It has been shown that significant muscular injuries result in areparative inflammatory-mediated healing response yielding fibrotic scarand dysfunctional muscle. For example, compromised laryngeal function,whether due to congenital malformations, trauma, cancer, or surgicaldefects, affects thousands of individuals worldwide each year¹.Unfortunately, therapeutic options to restore lost muscle and dynamiclaryngeal functions for these patients are limited. As a result,patients suffer devastating quality of life consequences, includingsevere voice impairment, an ineffective or unsafe swallow, or airwayobstruction, often-necessitating gastrostomy and/or tracheostomy tubes.Advanced tissue engineering and regenerative strategies aimed to developskeletal muscle implants may provide clinicians with new tools andtherapeutic strategies for treating these patients.

Normal skeletal muscle reveals an intricate tissue design, includingmuscle fibers with their associated contractile machinery and a richneurovascular supply which is essential for inducing and sustainingdynamic contraction. Furthermore, muscle extracellular matrix (ECM),which includes fibrillar type I collagen as a major component, plays animportant role in guiding the muscle-nerve-vascular interface as well assupporting muscle's mechanical function, adaptability, and repair⁴.Therefore, it is not surprising that the majority of muscle engineeringapproaches focus on capturing these essential design features. Atpresent, the most widely used cell source for engineering skeletalmuscle is the putative muscle progenitor cell (MPC; satellite cell),which can be readily isolated from muscle biopsies and cultured toproduce myoblasts⁵. MPC can be further modified to inducedifferentiation and expression of motor endplates, generating motorendplate expressing MPCs, referred to hereafter as MEE. In turn, thesecells are interfaced with a variety of natural and syntheticbiomaterials, designed to promote myoblast fusion, differentiation, andmaturation in vitro or in vivo.

To achieve meaningful therapeutic benefit, it has been proposed thatengineered muscle should i) be constructed from autologous cell sources,ii) recapitulate the structure and functional properties of nativeskeletal muscle, which represents aligned muscle fibers interfacingwithin an appropriate, well-organized (ECM), iii) integrate rapidly intohost tissue with associated neovascularization and innervation, and iv)support scalable and patient-specific design^(2,3).

Synthetic polymers, including polycaprolactone andpoly(lactic-co-glycolic) acid, often are the engineering material ofchoice, largely owing to their mechanical stability, design versatility,and amenability to “additive” micro- and nano-fabrication techniques(electrospinning, patterning), where cells and materials are broughttogether in a stepwise fashion by adding layer upon layer.Unfortunately, upon implantation in vivo, these materials are sensed as“foreign” to cells, yielding an inflammatory-mediated, foreign-bodyresponse which is known to compromise healing and lead to poor clinicaloutcomes. Decellularized tissues (e.g., skeletal muscle), which areprocessed to maintain the complex composition, structural integrity, andarchitectural features of tissue extracellular matrix (ECM), also havebeen applied. However, these graft materials induce an inflammatoryreaction as well, and their dense microstructure prevents completerecellularization and muscle recovery. Alternatively, natural polymers,such as fibrinogen, type I collagen, and Matrigel alone or incombination have been employed.

For these applications, conventional casting methods have been appliedwhere cells are mixed with natural polymers and pipetting within moldsto form cylindrical or rectangular shaped constructs with randomlyorganized dispersions of cells. The materials are then anchored at eachend to provide passive tension, which is required to promoteunidirectional cell alignment and myotube formation via cell fusion.Although it is evident that these natural polymers provide cell adhesionsites and associated bioinstructive properties, they are known toexhibit high batch-to-batch variability and are less amenable to thecontrol of their physicochemical properties and scalable fabricationprocesses than synthetic polymers. Despite advancements with respect tomuscle engineering, the search continues for a cost-effective andcustomizable muscle fabrication strategy that harnesses natural muscleformation processes (known as myogenesis) and rapid integration andneurovascular regeneration in absence of inflammation followingimplantation within the body.

Applications of such functional engineered muscle vary. For example,such muscle could be used to repair post-oncologic or traumatic defects,or to medialize the vocal fold in cases of paresis/paralysis.Autologous, organized, engineered muscle that has adequate bulk,integrates into host tissue, and restores tissue structure and functionin absence of inflammation does not currently exist. Therefore, there isan unmet need for advanced scalable manufacturing strategies forengineered biological compositions, such as collagen and tissuecompositions for tissue restoration in the presence, or in the absenceof, embedded cells.

SUMMARY OF THE INVENTION

In one aspect of the disclosure, kits for tissue reconstructioncomprising a polymerizable collagen solution and a syringe are provided.In an additional aspect of the disclosure, kits comprising a solidcollagen construct and a mold are provided.

In yet an additional aspect of the disclosure, solid collagen constructscomprising aligned collagen fibrils and aligned cells are provided. In astill further aspect of the disclosure, solid collagen constructsprepared by the process of extruding a polymerizable collagen solutionwith an extruder to generate solid collagen constructs are provided.

In yet an additional aspect of the disclosure, solid collagen constructsprepared by the process of extruding a suspension of polymerizablecollagen solution and cells with an extruder to generate solid collagenconstructs are provided. In a further aspect of the disclosure, tissueimplants comprising solid collagen constructs are provided.

In an additional aspect of the disclosure, processes for preparing solidcollagen constructs are provided comprising extruding a polymerizablecollagen solution with an extruder to generate solid collagen constructsare provided.

In still a further aspect of the disclosure, processes for preparingsolid collagen constructs by the process comprising extruding asuspension of polymerizable collagen solution and cells with an extruderto generate solid collagen constructs wherein the solid collagenconstructs are embedded with cells are provided.

In a further aspect of the disclosure, a 3D tissue-engineered muscleimplant prepared from therapeutic cells, and type I collagen oligomersthrough extrusion, is provided.

Still other embodiments described in the following clause list areconsidered to be part of the invention.

In addition any of the embodiments described in the following clauselist are considered to be part of the invention.

-   1. A solid collagen construct comprising aligned collagen fibrils    and aligned cells.-   2. A solid collagen construct prepared by the process of extruding a    polymerizable collagen solution with an extruder to generate the    solid collagen construct.-   3. The solid collagen construct of clause 2, wherein the    polymerizable collagen solution is a polymerizable collagen oligomer    solution.-   4. The solid collagen construct of clause 3, wherein the solution is    extruded into a container.-   5. The solid collagen of clause 4, wherein the container is at a    higher temperature than the extruder.-   6. The solid collagen construct of clause 5, wherein the extruder is    at about 4° C. and the container is at about 37° C.-   7. The solid collagen construct of clauses 2-6, wherein the extruder    is a syringe.-   8. The solid collagen construct of clauses 3-7, wherein the    polymerizable collagen oligomer solution comprises collagen    oligomer, water, an acid, one or more salts, and a base.-   9. The solid collagen construct of clause 8, wherein the    polymerizable collagen oligomer solution further comprises a sugar.-   10. The solid collagen construct of clauses 8-9, wherein the acid is    HCl and the base is NaOH.-   11. The solid collagen construct of clauses 8-10, wherein the one or    more salts are KH₂PO₄, Na₂HPO₄, KCl, and NaCl.-   12. The solid collagen construct of clauses 9-11, wherein the sugar    is glucose.-   13. The solid collagen construct of clauses 2-11, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 0.1 mg/ml and about 40 mg/ml.-   14. The solid collagen construct of clause 13, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 1 mg/ml and about 10 mg/ml.-   15. The solid collagen construct of clause 14, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 2 mg/ml and about 6 mg/ml.-   16. The solid collagen construct of clause 15, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 3 mg/ml and about 5 mg/ml.-   17. The solid collagen construct of clauses 2-16, wherein the    polymerizable collagen solution is extruded at a rate of between    about 1 ml/minute and about 3 ml/minute.-   18. The solid collagen construct of clause 16, wherein the    polymerizable collagen solution is extruded at a rate of about 2    ml/minute.-   19. The solid collagen construct of clauses 2-18, wherein the pH of    the polymerizable collagen solution is between about 4 and about 10.-   20. The solid collagen construct of clause 19, wherein the pH of the    polymerizable collagen solution is between about 6 and 8.-   21. The solid collagen construct of clause 20, wherein the pH of the    polymerizable collagen solution is about 7.4.-   22. The solid collagen construct of clauses 2-21, wherein the solid    collagen is fibrillar.-   23. The solid collagen construct of clauses 2-22, wherein the    polymerizable collagen solution polymerizes during extrusion in the    extruder.-   24. The solid collagen construct of clauses 2-23, wherein the    polymerizable collagen solution polymerizes after extrusion from the    extruder.-   25. The solid collagen construct of clauses 4-24 wherein the    container is a die or mold.-   26. The solid collagen construct of clause 26, wherein the container    is a mold.-   27. A solid collagen construct prepared by the process comprising    extruding a suspension of polymerizable collagen solution and cells    with an extruder to generate a solid collagen construct wherein the    solid collagen construct is embedded with cells.-   28. The solid collagen construct of clause 27, wherein the    polymerizable collagen solution is a polymerizable collagen oligomer    solution.-   29. The solid collagen construct of clauses 27-28, wherein the cells    are MPC cells.-   30. The solid collagen construct of clauses 27-28, wherein the cells    are MEE cells.-   31. The solid collagen construct of clauses 27-28, wherein the cells    are ASC.-   32. The solid collagen construct of clauses 27-31, wherein the    polymerizable collagen oligomer suspension is extruded into a    container.-   33. The solid collagen construct of clause 32, wherein the container    is at a higher temperature than the extruder.-   34. The solid collagen construct of clause 33, wherein the extruder    is at about 4° C. and the container is at about 37° C.-   35. The solid collagen construct of clauses 27-34, wherein the    extruder is a syringe.-   36. The solid collagen construct of clauses 28-35, wherein the    polymerizable oligomer solution comprises collagen oligomer, water,    an acid, one or more salts, and a base.-   37. The solid collagen construct of clauses 36, wherein the    polymerizable collagen oligomer solution further comprises a sugar.-   38. The solid collagen construct of clauses 36-37, wherein the acid    is HCl and the base is NaOH.-   39. The solid collagen construct of clauses 36-38, wherein the one    or more salts are KH₂PO₄, Na₂HPO₄, KCl, and NaCl.-   40. The solid collagen construct of clauses 37-39, wherein the sugar    is glucose.-   41. The solid collagen construct of clauses 27-40, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 0.1 mg/ml and about 40 mg/ml.-   42. The solid collagen construct of clause 41, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 1 mg/ml and about 6 mg/ml.-   43. The solid collagen construct of clause 42, wherein the    concentration of the collagen in the polymerizable collagen solution    is between about 3 mg/ml and about 5 mg/ml.-   44. The solid collagen construct of clauses 27-43, wherein the    polymerizable collagen suspension is extruded at a rate of between    about 1 ml/minute and about 3 ml/minute.-   45. The solid collagen construct of clause 44, wherein the    polymerizable collagen suspension is extruded at a rate of about 2    ml/minute.-   46. The solid collagen construct of clauses 27-45, wherein the pH of    the polymerizable collagen solution is between about 4 and about 10.-   47. The solid collagen construct of clause 46, wherein the pH of the    polymerizable collagen solution is between about 6 and 8.-   48. The solid collagen construct of clause 47, wherein the pH of the    polymerizable collagen solution is about 7.4.-   49. The solid collagen construct of clauses 27-48, wherein the solid    collagen is fibrillar.-   50. The solid collagen construct of clauses 27-49, wherein the    polymerizable collagen suspension polymerizes during extrusion in    the extruder.-   51. The solid collagen construct of clauses 27-50, wherein the    polymerizable collagen suspension polymerizes after extrusion from    the extruder.-   52. The solid collagen construct of clauses 32-51 wherein the    container is a die or mold.-   53. The solid collagen construct of clause 52, wherein the container    is a mold.-   54. The solid collagen construct of clauses 27-53 further comprising    the step of culturing the solid collagen construct.-   55. The solid collagen construct of clauses 27-54, wherein the solid    collagen construct is in the form of tissues with aligned    architectures.-   56. The solid collagen construct of clause 55, where the tissue is    selected from cardiac muscle, nerve, smooth muscle, tendon, and    ligament.-   57. The solid collagen construct of clause 56, wherein the tissue is    a muscle.-   58. The solid collagen construct of clause 57, wherein the muscle is    skeletal muscle, adductor muscle, cardiac muscle, or smooth muscle.-   59. Tissue implants for human or veterinary use comprising the solid    collagen constructs of clauses 2-58.-   60. Tissue implants of clause 59 wherein the tissue is selected from    cardiac muscle, nerve, smooth muscle, tendon, and ligament.-   61. The tissue implants of clause 60, wherein the tissue is a    muscle.-   62. The tissue implants of clause 61, wherein the muscle is skeletal    muscle, cardiac muscle. or smooth muscle.-   63. A process of preparing solid collagen constructs comprising    extruding a polymerizable collagen solution with an extruder to    generate a solid collagen construct.-   64. The process of clause 3, wherein the polymerizable collagen    solution is a polymerizable collagen oligomer solution.-   65. The process of clause 64, wherein the solution is extruded into    a container.-   66. The solid collagen of clause 65, wherein the container is at a    higher temperature than the extruder.-   67. The process of clause 66, wherein the extruder is at about 4° C.    and the container is at about 37° C.-   68. The process of clauses 63-67, wherein the extruder is a syringe.-   69. The process of clauses 64-68, wherein the polymerizable collagen    oligomer solution comprises collagen oligomer, water, an acid, one    or more salts, and a base.-   70. The process of clause 69, wherein the polymerizable collagen    oligomer solution further comprises a sugar.-   71. The process of clauses 69-70, wherein the acid is HCl and the    base is NaOH.-   72. The process of clauses 69-71, wherein the one or more salts are    KH₂PO₄, Na₂HPO₄, KCl, and NaCl.-   73. The process of clauses 70-71, wherein the sugar is glucose.-   74. The process of clauses 63-71, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 0.1    mg/ml and about 40 mg/ml.-   75. The process of clause 74, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 1    mg/ml and about 10 mg/ml.-   76. The process of clause 75, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 2    mg/ml and about 6 mg/ml.-   77. The process of clause 76, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 3    mg/ml and about 5 mg/ml.-   78. The process of clauses 63-77, wherein the polymerizable collagen    solution is extruded at a rate of between about 1 ml/minute and    about 3 ml/minute.-   79. The process of clause 77, wherein the polymerizable collagen    solution is extruded at a rate of about 2 ml/minute.-   80. The process of clauses 63-79, wherein the pH of the    polymerizable collagen solution is between about 4 and about 10.-   81. The process of clause 80, wherein the pH of the polymerizable    collagen solution is between about 6 and 8.-   82. The process of clause 81, wherein the pH of the polymerizable    collagen solution is about 7.4.-   83. The process of clauses 63-82, wherein the solid collagen is    fibrillar.-   84. The process of clauses 63-83, wherein the polymerizable collagen    solution polymerizes during extrusion in the extruder.-   85. The process of clauses 63-84, wherein the polymerizable collagen    solution polymerizes after extrusion from the extruder.-   86. The process of clauses 65-85 wherein the container is a die or    mold.-   87. The process composition of clause 86, wherein the container is a    mold.-   88. A process for preparing solid collagen construct comprising    extruding a suspension of polymerizable collagen solution and cells    with an extruder to generate a solid collagen construct wherein the    solid collagen construct is embedded with cells.-   89. The process of clause 88, wherein the polymerizable collagen    solution is a polymerizable collagen oligomer solution.-   90. The process of clauses 88-89, wherein the cells are MPC cells.-   91. The process of clauses 88-89, wherein the cells are MEE cells.-   92. The process of clauses 88-89, wherein the cells are ASC.-   93. The process of clauses 88-92, wherein the polymerizable collagen    oligomer suspension is extruded into a container.-   94. The process of clause 93, wherein the container is at a higher    temperature than the extruder.-   95. The process of clause 94, wherein the extruder is at about 4° C.    and the container is at about 37° C.-   96. The process of clauses 88-95, wherein the extruder is a syringe.-   97. The process of clauses 89-96, wherein the polymerizable oligomer    solution comprises collagen oligomer, water, an acid, one or more    salts, and a base.-   98. The process of clauses 97, wherein the polymerizable collagen    oligomer solution further comprises a sugar.-   99. The process of clauses 97-98, wherein the acid is HCl and the    base is NaOH.-   100. The process of clauses 97-99, wherein the one or more salts are    KH₂PO₄, Na₂HPO₄, KCl, and NaCl.-   101. The process of clauses 98-100, wherein the sugar is glucose.-   102. The process of clauses 88-101, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 1    mg/ml and about 10 mg/ml and the solid collagen construct forms    fibrils.-   103. The process of clause 102, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 2    mg/ml and about 6 mg/ml.-   104. The process of clause 103, wherein the concentration of the    collagen in the polymerizable collagen solution is between about 3    mg/ml and about 5 mg/ml.-   105. The process of clauses 88-104, wherein the polymerizable    collagen suspension is extruded at a rate of between about 1    ml/minute and about 3 ml/minute.-   106. The process of clause 105, wherein the polymerizable collagen    suspension is extruded at a rate of about 2 ml/minute.-   107. The process of clauses 88-106, wherein the pH of the    polymerizable collagen solution is between about 4 and about 10.-   108. The process of clause 107, wherein the pH of the polymerizable    collagen solution is between about 6 and 8.-   109. The process of clause 108, wherein the pH of the polymerizable    collagen solution is about 7.4.-   110. The process of clauses 88-109, wherein the solid collagen is    fibrillar.-   111. The process of clauses 88-110, wherein the polymerizable    collagen suspension polymerizes during extrusion in the extruder.-   112. The process of clauses 88-111, wherein the polymerizable    collagen suspension polymerizes after extrusion from the extruder.-   113. The solid collagen composition of clauses 93-112 wherein the    container is a die or mold.-   114. The solid collagen composition of clause 113, wherein the    container is a mold.-   115. The process of clauses 88-114, further comprising the step of    culturing the solid collagen construct.-   116. The process of clauses 88-115, wherein the solid collagen    construct is in the form of tissues with aligned architectures.-   117. The process of clause 116, where the tissue is selected from    cardiac muscle, nerve, smooth muscle, tendon, and ligament.-   118. The process of clause 117, wherein the tissue is a muscle.-   119. The process of clause 118, wherein the muscle is skeletal    muscle, cardiac muscle, or smooth muscle.-   120. The solid collagen compositions of clause 41, wherein the    concentration of collagen in the polymerizable collagen solution is    between about 1 mg/ml and about 10 mg/ml.-   121. The solid collagen composition of clauses 24 and 51, wherein    polymerization occurs after extrusion from the extruder.-   122. The process of clauses 85 and 112 wherein polymerization occurs    after extrusion from the extruder.-   123. A kit for tissue reconstruction comprising a polymerizable    collagen solution or a polymerizable collagen suspension and a    syringe.-   124. The kit of clause 123, wherein the polymerizable collagen    solution is a solution of polymerizable collagen oligomers.-   125. A kit for tissue reconstruction comprising a solid collagen    construct and a mold.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic showing extrusion of polymerizable type I collagenoligomer suspension containing cells through a temperature-controlledmold for rapid production of engineered tissue constructs. Duringextrusion the soluble oligomer molecules undergo thermally controlledpolymerization to form oriented solid fibrils which further associate toform aligned composites comprised of solid fibrillar collagen and cells.Extrusion can also be done in the absence of cells.

FIG. 2: Examples of extrusion mold geometries used to create engineeredcollagen and tissue constructs.

FIG. 3. Representative images and directionality analyses for engineeredmuscle constructs formed using extrusion method (A-C) compared toconventional casting technique (D-F). Fabricated muscle constructs werecultured 24 hours and analyzed by confocal reflection microscopy forvisualization of fibril microstructure (A,D), confocal fluorescencemicroscopy for visualization of myoblast morphology (B,E), and ImageJDirectionality algorithm for alignment determination (C,F). Scale bar=50μm

FIG. 4A. Engineered muscle construct culture and optimization. Muscleconstructs were formed by extrusion of C2C12 myoblasts at 10E6 cells/mL(a,b) or 10E7 cells/mL (c,d) suspended within type I collagen oligomersolutions prepared at 1.3 mg/mL (a,c) and 3.3 mg/mL (b,d). A. Constructswere cultured within a custom culture device for 14 days under passivetension.

FIG. 4B. Confocal images (100 μm thickness) of 14-day constructs stainedwith phalloidin (F-actin) and Draq5 (nucleus) show more extensivemyotube formation (white arrows) for constructs prepared at high celldensities (10E7 cells/mL) and low oligomer concentrations (1.3 mg/mL).Scale bar=50 μm

FIG. 5. Engineered muscle constructs (14 days) formed by extrusion ofF344 rat primary MPCs (10E7 cells/mL) suspended within type I collagenoligomer solutions prepared at 1.3 mg/mL. A. Confocal images (50 μmthickness) of 14-day constructs stained with phalloidin (F-actin) andDraq5 (nucleus) show highly aligned cell morphology and fused myotubeformation. Cells were distributed throughout the entire construct volumeas indicated by H&E staining of longitudinal (B) and transverse (C)sections. Scale bars=30 μm (A), 500 μm (B-C)

FIG. 6: Representative images and directionality analyses for engineeredtissue constructs formed with adipose-derived stem cells (ASCs)suspended in type I collagen oligomer solution using extrusion method(A-B) compared to conventional casting technique (C-D). Fabricatedconstructs were cultured 24 hours, stained with phalloidin (F-actin) andDraq5 (nucleus) and analyzed by confocal fluorescence microscopy forvisualization of cell morphology (A,C) and ImageJ Directionalityalgorithm for alignment determination (B,D). Scale bars=100 μm.

FIG. 7: Representative confocal reflection microscopy image ofengineered collagen construct with aligned collagen fibrils formed byextrusion of a type I collagen oligomer solution prepared at aconcentration of 3.3 mg/mL. Scale bar=10 μm.

FIG. 8. (A) Schematic showing partial laryngectomy model, which involvedremoval of a section of hemi-thyroid cartilage and associated adductormuscle from left side. Cross-section of rat larynx showing surgicalprocedure and implant placement. (B-E) Representative H&E stainedsections of untreated control defect (B, 2 months) and defects treatedwith oligomer-only implant (C, 2 months), engineered muscle implant (D,1 month), and engineered muscle implant (E, 3 months). Untreated defect(B) showed gap filled with native sternohyoid muscle and healing viainflammation and fibrotic scar tissue formation. Oligomer-only groupshowed no significant inflammatory response and construct populated withmesenchymal cells. Engineered muscle implants showed progressiveincrease in volume of striated muscle (boxed areas and inserts; insertsrepresent high magnification of boxed areas) and cartilage regeneration(arrow) over time with similar responses observed for both MPC- andMEE-oligomer implants). Asterix (*) indicate edges of cartilage defect.Scale bar=200 μm

FIG. 9. Representative electromyography (EMG) tracings captured at 50 μVamplitude and 10 ms sweep speeds during active laryngospasm to detectfiring within engineered muscle implant 3 months following implantation(A,B) or native adductor muscle complex (C). A. Defect treated withengineered muscle prepared with MPCs (MPC Muscle) generated abundant,variable-sized motor unit potentials. B. Engineered muscle prepared withmotor endplate expressing MPCs (MEE Muscle) generated potentials thatwere significantly larger in amplitude and frequency compared to MPCMuscle and native adductor muscle. C. Native adductor muscle complexdemonstrated bursts of motor unit potentials during laryngospasm.

FIG. 10. Anti-beta III tubulin and α-bungarotoxin staining of (A)untreated control defect, (B) oligomer-only control, (C) MSC-oligomerconstruct, and (D) MEE-oligomer construct at 3 months following surgicalimplantation post-op. Dotted line outlines approximate constructboundaries. The MEE-oligomer construct (D) showed increased innervationwhen compared to the MEE-oligomer construct (C). Both the MEE- andMSC-oligomer constructs exhibited increased innervation when compared tothe oligomer-only and untreated controls. Scale bar=100 μm

FIG. 11: (A) Schematic showing partial laryngectomy model, whichinvolved removal of a section of hemi-thyroid cartilage and associatedunderlying adductor muscle in the presence or absence of recurrentlaryngeal nerve injury. Cross-section of rat larynx showing surgicalreconstruction with tissue-engineered cartilage and muscle implants. Thecartilage constructs showed progressive remodeling, increasedproteoglycan deposition, and maturation over the 1, 3, and 6 monthtimepoints (B, C, D respectively). Asterix represents edges ofsurgically created defect. Scale Bar=100 μm

FIG. 12: H&E staining shows progressive restoration of partiallaryngectomy defect reconstructed with engineered muscle constructs at 1month (A,D), 3 months (B,E), and 6 months (C,F), marked by muscularintegration, regeneration, and maturation with no inflammatory response.Asterix marks edges of surgical defect. Scale Bar: A-C 100 μm; D-F 25 μm

FIG. 13: α-bungarotoxin and beta III tubulin staining shows presence ofmotor endplates and significant innervation of the engineered muscleconstructs after 6 months. A: MSC-oligomer constructs showed a lesserdegree of innervation B. MEE-oligomer constructs showed robustinnervation and motor endplate expression (arrowheads). Scale Bar=10 μm

FIG. 14A: Animals with injured recurrent laryngeal nerves (RLN) treatedwith MEE-oligomer constructs showed increased EMG activity and myofiberdiameter compared to MPC-oligomer implants. EMG of the adductor musclecomplex showed increased activity in MEE-oligomer (C) treated animalscompared to MPC-oligomer (A) treated animals. Histology showed somemuscle atrophy in MPC-oligomer (B) treated animals and near normalmyofiber architecture in MEE-oligomer (D) treated animals. Myofiberdiameter of adductor muscle (E) showed significant increase inMEE-oligomer treated animals (Mean+/−SD, p<0.001, 2 animals per group,minimum 36 measurements per animal). Scale bar=100 μm.

FIG. 14B: Bar graph illustrating mean myofiber diameter of adductormuscle following hemilaryngeal reconstruction within RLN injured ratswith MPC-oligomer constructs or MEE-oligomer constructs.

FIG. 15: Still images from video laryngoscopy obtained 3 months afterhemilaryngeal reconstruction in rat laryngectomy model with recurrentlaryngeal nerve injury. Recovery of left vocal fold movement wasobserved in animals treated with MEE-oligomer constructs (A). Animalstreated with the MSC-oligomer constructs (B) showed less pronouncedmovement in the left vocal fold.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

Unless defined otherwise, the scientific and technology nomenclatureshave the same meaning as commonly understood by a person in the ordinaryskill in the art pertaining to this disclosure.

In many embodiments of the disclosure, solid collagen constructs, andprocesses for making them, are provided wherein the solid collagenconstructs are prepared by extruding a polymerizable collagen solutionwith an extruder thereby generating a solid collagen construct. Thepolymerizable collagen solution is often a solution wherein the collagencontains oligomers such that the polymerizable collagen solution is anpolymerizable solution of oligomeric collagen. In many embodiments, thecollagen is only or primarily oligomeric collagen and thus contains noor substantially no monomeric collagen. The extrusion is often done intoa container such as a dye or mold. Often the extrusion is done in anextruder, such as a syringe, which is kept on ice (e.g., at about 4°C.). Although the polymerizable solution may polymerize at suchtemperatures, the polymerization conditions are kept such that extrusionis still possible and practical. When extruded into a container such asa mold or die, the container temperature is often kept at elevatedtemperatures whether room temperature or body temperature (e.g., 37° C.)to accelerate polymerization.

The polymerizable solution includes or contains components such thatpolymerization can be initiated at 4 C and accelerated at highertemperatures. For example, a typical polymerizable collagen oligomersolution may be prepared in accordance with Example 1. In manyembodiments, the polymerizable collagen oligomer solution containscollagen oligomers, such as a type 1 collagen oligomer, which has beendissolved in water and acid, such as HCl. The pH is raised in thepresence of one or more salts and base, such as NaOH. A sugar mayoptionally be added, such as glucose. An example of a combination ofsalts which is the one or more salts is KH₂PO₄, Na₂HPO₄, KCl, and NaCl.For example, when a solubilized collagen oligomeric solution is acidicand is then neutralized by the one or more salts and the base such thatthe pH increases to the range of between about 4 and 10 including withinabout 6 and about 8, and further including about 7.4, polymerizationspontaneously occurs with the rate of such polymerization beingdependent upon temperature.

Unless explicitly defined otherwise, the term, “solid collagenconstruct,” refers to collagen compositions made, for example, byextrusion, in accordance with the disclosure.

Some materials that may be used to practice some embodiments of theinvention can be found in U.S. Pat. No. 9,878,071 B2 issued on Jan. 30,2018 and incorporated fully herein by reference.

In some embodiments, the polymerizable collagen composition, which maybe a solution or a suspension, for example may be prepared under variousconditions. For example, factors such as pH, phosphate concentration,temperature, buffer composition, ionic strength, and composition andconcentration of the extracellular matrix components which includes bothcollagen and non-collagenous molecules, may be varied by additives orchanging the environmental conditions of the polymerizable collagencomposition. Examples of additives include nutrients, includingminerals, amino acids, sugars, peptides, proteins, vitamins (such asascorbic acid), or glycoproteins that facilitate hematopoietic stem cellculture, such as laminin and fibronectin, hyaluronic acid, or growthfactors such as platelet-derived growth factor, or transforming growthfactor beta, and glucocorticoids such as dexamethasone. Other additivesinclude fibrillogenesis inhibitors, such as glycerol, glucose, orpolyhydroxylated compounds can be added prior to or duringpolymerization. Additional additives include cross-linking agents, suchas carbodiimides, aldehydes, lysl-oxidase, N-hydroxysuccinimide esters,imidoesters, hydrazides, and maleimides, and the like can be addedbefore, during, or after polymerization.

With regards to sourcing the collagen starting material, it may besolubilized from tissue. For example, the collagen can be prepared byutilizing acid-solubilized collagen and defined polymerizationconditions that are controlled to yield three-dimensional collagenmatrices with a range of controlled assembly kinetics (e.g.,polymerization half-time), molecular compositions, and fibrilmicrostructure-mechanical properties, for example, as described in U.S.patent application Ser. No. 11/435,635 (published Nov. 22, 2007, asPublication No. 2007-0269476 A1) and Ser. No. 11/903,326 (published Oct.30, 2008, as Publication No. 2008-0268052), each incorporated herein byreference in its entirety. In other embodiments, the collagen ispolymerizable collagen. In yet other embodiment, the collagen is Type Icollagen.

In some embodiments, the sourced collagen starting material is unnaturalcollagen. As used herein, the phrase “unnatural collagen” refers tocollagen that has been removed from a source tissue. Optionally, theunnatural collagen may be solubilized from the tissue source. In otherembodiments, the collagen is synthetic collagen. In yet otherembodiments, the collagen is recombinant collagen.

In one aspect, unnatural collagen or collagen components can be used andcan be obtained from a number of sources, including for example, porcineskin, to construct the collagen compositions described herein. Suitabletissues useful as a collagen-containing source material for isolatingcollagen or collagen components to make the collagen compositionsdescribed herein are submucosa tissues or any other extracellularmatrix-containing tissues of a warm-blooded vertebrate. Suitable methodsof preparing submucosa tissues are described in U.S. Pat. Nos.4,902,508; 5,281,422; and 5,275,826, each incorporated herein byreference. Extracellular matrix material-containing tissues other thansubmucosa tissue may be used to obtain collagen in accordance with themethods and compositions described herein. Methods of preparing otherextracellular matrix material-derived tissues for use in obtainingpurified collagen or partially purified extracellular matrix componentsare known to those skilled in the art. For example, see U.S. Pat. No.5,163,955 (pericardial tissue); U.S. Pat. No. 5,554,389 (urinary bladdersubmucosa tissue); U.S. Pat. No. 6,099,567 (stomach submucosa tissue);U.S. Pat. No. 6,576,265 (extracellular matrix tissues generally); U.S.Pat. No. 6,793,939 (liver basement membrane tissues); and U.S. patentapplication publication no. US-2005-0019419-A1 (liver basement membranetissues); and international publication no. WO 2001/45765 (extracellularmatrix tissues generally), each incorporated herein by reference. Invarious other embodiments, the collagen-containing source material canbe selected from the group consisting of placental tissue, ovariantissue, uterine tissue, animal tail tissue, and skin tissue. In someembodiments, the collagen is selected from the group consisting of pigskin collagen, bovine collagen, and human collagen. Any suitableextracellular matrix-containing tissue can be used as acollagen-containing source material to isolate purified collagen orpartially purified extracellular matrix components.

An illustrative preparation method for preparing submucosa tissues as asource of purified collagen or partially purified extracellular matrixcomponents is described in U.S. Pat. No. 4,902,508, the disclosure ofwhich is incorporated herein by reference. In one embodiment, a segmentof vertebrate intestine, for example, preferably harvested from porcine,ovine or bovine species, but not excluding other species, is subjectedto abrasion using a longitudinal wiping motion to remove cells orcell-removal is accomplished by hypotonic or hypertonic lysis. In oneembodiment, the submucosa tissue is rinsed under hypotonic conditions,such as with water or with saline under hypotonic conditions and isoptionally sterilized. In another illustrative embodiment, suchcompositions can be prepared by mechanically removing the luminalportion of the tunica mucosa and the external muscle layers and/orlysing resident cells with hypotonic or hypertonic washes, such as withwater or saline. In these embodiments, the submucosa tissue can bestored in a hydrated or dehydrated state prior to isolation of thepurified collagen or partially purified extracellular matrix components.In various aspects, the submucosa tissue can comprise any delaminationembodiment, including the tunica submucosa delaminated from both thetunica muscularis and at least the luminal portion of the tunica mucosaof a warm-blooded vertebrate.

In some embodiments, the collagen is oligomeric collagen. The presenceof oligomeric collagen enhances the self-assembly potential byincreasing the assembly rate and by yielding collagen compositions withdistinct fibril microstructures and increased mechanical integrity(e.g., stiffness). In some embodiments, the collagen comprisesoligomeric collagen. In other embodiments, the collagen consistsessentially of oligomeric collagen. In yet other embodiments, thecollagen consists of oligomeric collagen.

In some embodiments, the collagen is monomeric collagen. In someembodiments, the collagen is atelocollagen. As used herein, the term“atelocollagen” refers to collagen that is treated in vitro with pepsinor another suitable protease or agent to eliminate or substantiallyreduce telopeptide regions which contain intermolecular cross-linkingsites. In other embodiments, the monomeric collagen is telocollagen. Asused herein, the term “telocollagen” refers to acid solubilized collagenthat retains its telopeptide ends.

In certain embodiments, the collagen comprises oligomeric collagen andatelocollagen. In other embodiments, the collagen comprises oligomericcollagen, monomeric collagen, and atelocollagen. The amounts ofoligomeric collagen, monomeric collagen, and/or atelocollagen may beformulated in the collagen compositions to advantageously maximize thestiffness, strength, fluid and mass transport, proteolytic degradationor compatibility of the engineered collagen compositions.

In any of the embodiments described herein, the collagen can have apredetermined percentage of collagen oligomers. In various embodiments,the predetermined percentage of collagen oligomers can be about 0.5% toabout 100%, about 30% to about 100%, about 40% to about 100%, about 50%to about 100%, about 60% to about 100%, about 70% to about 100%, about80% to about 100%, about 90% to about 100%, about 95% to about 100%, orabout 100%. In yet another embodiment, the collagen oligomers areobtained from a collagen-containing source material enriched withcollagen oligomers (e.g., pig skin).

In any of the embodiments described herein, the collagen sourced asstarting material can have an oligomer content quantified by averagepolymer molecular weight (AMW). As described herein, modulation of AMWcan affect polymerization kinetics, fibril microstructure, molecularproperties, and fibril architecture of the matrices, for example,interfibril branching, pore size, and mechanical integrity (e.g., matrixstiffness). In another embodiment, the oligomer content of the purifiedcollagen, as quantified by average polymer molecular weight, positivelycorrelates with matrix stiffness.

In some embodiments, the collagen is reduced collagen. As used herein“reduced collagen” means collagen that is reduced in vitro to eliminateor substantially reduce reactive aldehydes. For example, collagen may bereduced in vitro by treatment of collagen with a reducing agent (e.g.,sodium borohydride).

In some embodiments, the collagen is oligomer 260 collagen. As usedherein “oligomer 260 collagen” is a collagen preparation made (e.g.,from porcine skin), by procedures resulting in isolation of oligomers,where the collagen preparation has a prominent band at molecular weight260, where the band is not prominent or is lacking in correspondingmonomer preparations. The presence of the band can be determined by SDSpolyacrylamide gel electrophoresis. Oligomer 260 collagen is furtherdescribed U.S. patent application Ser. No. 13/192,276 (published Feb. 2,2012, as Publication No. 2012-0027732 A1), incorporated herein byreference.

The solid collagen constructs herein described may be made undercontrolled conditions, such as by extrusion, and by changing extrusionparameters such as, for example, extrusion rate, geometry of thecontainer (e.g., mold), viscosity of polymerizable solution orsuspension, presence or absence of additives, cell density, temperature,porosity of extrusion container (e.g., mold) to obtain particularphysical properties. For example, the solid collagen constructs may havedesired collagen fibril density, pore size (fibril-fibril branching),elastic modulus, tensile strain, tensile stress, linear modulus,compressive modulus, loss modulus, fibril area fraction, fibril volumefraction, collagen concentration, cell seeding density, shear storagemodulus (G′ or elastic (solid-like) behavior), and phase angle delta(.delta. or the measure of the fluid (viscous)—to solid (elastic)—likebehavior; .delta. equals 0.degree. for Hookean solid and 90.degree. forNewtonian fluid).

As used herein, a “modulus” can be an elastic or linear modulus (definedby the slope of the linear region of the stress-strain curve obtainedusing conventional mechanical testing protocols; i.e., stiffness), acompressive modulus, a loss modulus, or a shear storage modulus (e.g., astorage modulus). These terms are well-known to those skilled in theart.

As used herein, a “fibril volume fraction” (i.e., fibril density) isdefined as the percent area of the total area occupied by fibrils inthree dimensions.

In any embodiment described herein, the fibril volume fraction of thecollagen composition is about 1% to about 60%. In various embodiments,the collagen composition can contain fibrils with specificcharacteristics, for example, a fibril volume fraction (i.e., density)of about 2% to about 60%, about 2% to about 40%, about 5% to about 60%,about 15% to about 60%, about 2% to about 30%, about 5% to about 30%,about 15% to about 30%, or about 20% to about 30%.

It may be desirable to control or identify the concentration of thecollagen, such as the collagen oligomer in solution, since differentconcentrations may yield solid collagen constructs with differentproperties. Typical ranges of collagen concentrations in thepolymerizable collagen solutions, such as polymerizable oligomercollagen solutions, include between about 0.1 mg/ml and about 40 mg/ml,including between about 1 mg/ml and about 10 mg/ml, including betweenabout 2 mg/ml and about 6 mg/ml, including between about 3 mg/ml andabout 5 mg/ml. The rate of extrusion through the extruder such as, forexample, a syringe, may also be controlled. Exemplary rates includebetween about 1 ml/min and about 3 ml/min including, or example, about 2ml/min.

When the solid construct polymerizes, whether in the syringe duringextrusion, or after exiting the extruder such as in a container (such asa die or mold), or both, the solid collagen construct may be in the formof fibrils. Such fibrils may be aligned due to the extrusion processsuch as for example, as see in FIG. 1 and FIG. 7. Such alignment can bebeneficial in the preparation of implants for tissues that have similaralignment geometries. In these and other embodiments of the invention,solid collagen constructs, and processes for preparing them, areprovided by extruding a suspension comprising a polymerizable collagensolution with cells, such as a polymerizable collagen oligomer solution,with an extruder to generate a solid collagen construct wherein thesolid collagen construct is embedded with cells. Example of such cellsinclude MCC, MEE, and/or ASC cells. In such embodiments, the suspensionmay be extruded into a container such as a die or a mold. Thetemperature of the extruder may be kept on ice (e.g., at about 4° C.)and the container, such as the die or mold, at room temperature or bodytemperature (e.g., at about 37° C.) to accelerate polymerization. Theextruder may be a syringe. The polymerizable solution containscomponents such that polymerization can be initiated at 4° C. andaccelerated at higher temperatures. For example, a typical polymerizablecollagen oligomer solution may be prepared in accordance with Example 1.In many embodiments, the polymerizable collagen oligomer solutioncontains collagen oligomers, such as a type 1 collagen oligomer, whichhas been dissolved in water and acid, such as HCl. The pH is raised inthe presence of one or more salts and base, such as NaOH. A sugar mayoptionally be added, such as glucose. An example of a combination ofsalts which is the one or more salts is KH₂PO₄, Na₂HPO₄, KCl, and NaCl.For example, when a solubilized collagen oligomeric solution is acidicand is then neutralized by the one or more salts and the base such thatthe pH increases to the range of between about 4 and 10 including withinabout 6 and about 8, and further including about 7.4, polymerizationspontaneously occurs with the rate of such polymerization beingdependent upon temperature.

It may be desirable to control or identify the concentration of thecollagen, such as the collagen oligomer in solution, since differentconcentrations may yield solid collagen constructs with differentproperties. Typical ranges of collagen concentrations in thepolymerizable collagen solutions, such as polymerizable oligomercollagen solutions, include between about 0.1 mg/ml and about 40 mg/ml,including between about 1 mg/ml and about 10 mg/ml, including betweenabout 2 mg/ml and about 6 mg/ml, including between about 3 mg/ml andabout 5 mg/ml.

The rate of extrusion of the polymerizable collagen suspension throughthe extruder such as, for example, a syringe, may also be controlled.Exemplary rates include between about 1 ml/min and about 3 ml/minincluding, or example, about 2 ml/min. When the solid constructpolymerizes, whether the suspension is in the syringe during extrusion,or after exiting the extruder such as in a container (such as a die ormold), or both, the solid collagen construct may be in the form offibrils. Such fibrils may be aligned due to the extrusion process suchas for example, as see in FIG. 1 and FIG. 7. Such alignment can bebeneficial in the preparation of implants for tissues that have similaralignment geometries. In many embodiments, the solid collagen constructswith embedded cells may further be cultured.

The solid collagen constructs embedded with cells may be used to formtissues with aligned architectures such as muscles, nerves, tendons, orligaments. Examples of muscle tissue include cardiac muscle, smoothmuscle, skeletal muscle, and adductor muscle. Collagen constructs madewith or without embedded cells may be used as implants in human orveterinary applications such as in tissues form tissues with alignedarchitectures such as muscles, nerves, tendons, or ligaments. Examplesof muscle tissue include cardiac muscle, smooth muscle, skeletal muscle,and adductor muscle. The kits of the disclosure may containpolymerizable collagen solutions or polymerizable collagen suspension,such as from oligomeric collagen, provided that the rate ofpolymerization is low enough to allow for extrusion. Other kits maycomprise solid collagen constructs. Such kits may be used for tissuereconstruction.

In many embodiments, the processes of the disclosure for making involvesextrusion of polymerizable collagen solutions. Polymerization describesthe process by which soluble collagen molecules aggregate to form solidcollagen fibrils surrounded by a fluid. For example, extrusion ofpolymerizable collagen solutions during polymerization yieldscompositions where solid collagen fibrils are preferentially oriented inthe direction of flow. Extrusion parameters, including temperature,oligomer concentration, flow rate, etc. can be modulated to yielddifferent compositions. Autologous stem, progenitor, or differentiatedcells and oligomer collagen for generation of different tissuecompositions. For example, tissue constructs that exhibit the greatestin-vitro muscle forming activity, as measured by the extent of myoblastfusion and myotube formation, is observed with the extrusion of anappropriate high density cells cultured within a low fibril density(stiffness) matrix formed from soluble collagen oligomers. In yetanother example, engineered muscle constructs can be generated bycombining oligomer collagen with myoblasts cell lines, muscle progenitorcells (MPC) (for example, autologous MPCs), or motor endplate expressingMPCs (MEE). For instance, constructs fashioned from oligomers and motorendplate expressing MPCs (MEE) provide advantageous in-vivo tissueintegration and muscle regeneration compared to MPC-oligomer constructs.As used herein, term “constructs” refers to solid collagen materialswither with or without embedded cells.

In other embodiments, solid collagen constructs such as alignedASC-oligomer or fibrillar collagen constructs are formed by extrusionthrough a temperature-controlled mold, and applied to polymerizableoligomer solutions during polymerization in the presence of cells in thecase of ASC-oligomer constructs or in the absence of cells in the caseof fibrillar collagen constructs.

In other embodiments, 3D tissue-engineered muscle implant prepared fromtherapeutic cells, and type I collagen oligomers through extrusion, isprovided. Examples of therapeutic cells including stem or progenitorcells (for example MPCs) or their derivatives (for example MEE MPCs). Inthese and other embodiments, 3D-Engineered muscle implants may beconstructed from autologous cell sources and polymerizable collagenoligomers to avoid adverse immune and inflammatory responses. Suchimplants recapitulate the structure and functional properties of nativeskeletal muscle, and represents aligned muscle fibers interfacing withinan appropriate, well-organized and persistent extracellular matrix (ECM)that shows low turnover rate or high resistance to proteolyticdegradation. The biological constructs integrate rapidly into hosttissue in absence of immune or inflammatory response with associatedneovascularization and innervation, interfacial tissue regeneration, andsupport scalable and patient-specific design. The ratio of cell densityand the oligomer fibril density may be optimized to achieve cell-matrixphysical and biochemical associations that recreate those found betweenmuscle cells and the endomysium in vivo, resulting in acceleratedin-vitro myotube formation and in-vivo muscle regeneration. For example,such solid collagen constructs made according to the disclosure can beused for reconstructing damaged muscle and cartilaginous hemilaryngealdefects as well as other muscle defects, which may result from a numberof conditions including traumatic injury, tumor extraction, muscledegeneration, myopathy, and congenital malformations.

In various embodiments of the invention, the myogenic potential of MPCsmay be interfaced with polymerizable collagens to create an engineeredmuscle for laryngeal reconstruction in the presence or in the absence ofrecurrent laryngeal nerve injury. For example, a MPC-oligomer constructmay be extruded to achieve a fibril density that mimics those foundbetween muscle cells and the endomysium in vivo, resulting inaccelerated in-vitro myotube formation. Additional benefit ofrecapitulating the muscle-ECM interface was evident from thetime-dependent recovery of muscle volume and function along withregeneration of supporting cartilaginous structures followingimplantation in vivo. Thus, for example, the advantage of the constructsof the disclosure is that they integrate with the surrounding normaltissue and immediately induce functional muscle formation within defectsites and do not trigger an inflammation response. Conventional muscleengineering strategies, which involve synthetic and naturalbiomaterials, are designed to degrade slowly so that they can bereplaced by host deposited tissue. However, the ability of the host togenerate new muscle in this situation in limited, especially since thesematerials typically induce inflammation.

The constructs described herein enjoy many advantages over the prior artengineered muscle tissue including i) the use of a standardized andcustomizable polymerizable collagen formulations, ii) the promotion ofinterfacial tissue regeneration by minimizing inflammation and iii)acceleration of functional muscle restoration through rapid innervation.For example, laryngeal reconstruction represents an unmet need becausethe methods to date are not suitable to perform such reconstructions.These prior methods have various limitations such as the lack of newmuscle formation, limited vascularization and innvervation which isrequired for functional muscle as well as triggering inflammation whichdelays healing. Currently, clinical strategies result in scar formationand loss of normal structure and function. By comparison, the methodsand materials of the disclosure involve engineered biological muscle,prepared from solid collagen constructs embedded with cells, overcomesthese limitations.

In many embodiments, and with respect to patient-specific humanlaryngeal muscle, the solid collagen constructs herein feature i)alignment of component MPCs and a collagen-fibril ECM rapidly uponfabrication (for example, via extrusion methods) and ii) induction ofmotor endplate expression to accelerate innervation following in-vivoimplantation⁷. The oligomers used within the constructs are well suitedfor tissue engineering and regeneration strategies since they i) exhibitrapid suprafibrillar self-assembly yielding highly interconnectedcollagen-fibril matrices resembling those found in vivo; ii) arestandardized based upon their fibril-forming capacity; support cellencapsulation and distribution throughout the construct; and iv) allowcustomized multi-scale design across the broadest range of tissuearchitectures and physical properties.

SOME REPRESENTATIVE EXAMPLES Example 1: Preparation of CollagenComposition

Type I collagen oligomers were derived from the dermis of closed herdpigs and prepared as described previously (Bailey J L, Critser P J,Whittington C, Kuske J L, Yoder M C, Voytik-Harbin S L; Collagenoligomers modulate physical and biological properties ofthree-dimensional self-assembled matrices, Biopolymers (2011)95(2):77-93 and Kreger S T, Bell B J, Bailey J, Stites E, Kuske J,Waisner B, Voytik-Harbin S L; Polymerization and matrix physicalproperties as important design considerations for soluble collagenformulations, Biopolymers (2010) 93(8):690-707, both incorporated hereinby reference). Prior to use, lyophilized collagen oligomers weresolubilized in 0.01 N hydrochloric acid and rendered aseptic bychloroform exposure at 4° C. or sterile filtration using a 0.22 μmfilter. A Sirius Red (Direct Red 80) assay was used to determinecollagen concentration. Oligomer solutions were standardized based uponpurity as well as polymerization capacity according to the ASTMinternational consensus standard F3089-14 (ASTM Standard F3089, 2014,“Standard Guide for Characterization and Standardization ofPolymerizable Collagen-Based Products and Associated Collagen-CellInteractions”, ASTM International, West Conshohocken, Pa., F3089-14,www.astm.org). Polymerization capacity is defined by matrix shearstorage modulus (G′) as a function of oligomer concentration of thepolymerization reaction. In this way, a predictive formulary can beestablished that relates the concentration of a polymerizable oligomersolution to specific viscoelastic properties, namely shear storagemodulus, of the resultant polymerized oligomer scaffold. Polymerizationis induced using single-step neutralization with a 10× self-assemblyreagent (added at a ratio of 1 part to 9 parts acidic oligomer solution)prepared according to the following recipe:

2 g KH₂PO₄ (FW 136.09)11.5 g Na₂HPO₄ (FW 141.96)

2 g KCl (FW 74.55)

10 g glucose

80 g NaCl (FW 58.44) 20 ml 5N NaOH

It should be noted that the rate of polymerization is temperaturedependent, increasing, for example over the range of 4° C. and 37° C.

Example 2: Viscoelastic Properties Testing

Viscoelastic properties of polymerized oligomer constructs weredetermined using oscillatory shear mode on an AR2000 rheometer (TAInstruments, New Castle, Del.) as previously described (Kreger et al.,2010). Samples were polymerized on the rheometer stage for 30 minfollowed by a shear-strain sweep from 0.1% to 4% strain at 1 Hz. Theshear storage modulus (G′) at 1% strain was used as a measure ofscaffold mechanical integrity.

Example 3: Myoblast Cell Line and Primary MPCs

C2C12 mouse myoblasts (ATCC, Rockville, Md.) were cultured in Dulbecco'sModified Eagle Medium (DMEM; Fisher Scientific, Chicago, Ill.)supplemented with 1% penicillin, streptomycin, amphotericin B (PSF-1;HyClone, Logan, Utah), and 10% fetal bovine serum (HyClone; Logan Utah)at 37° C. and 5% carbon dioxide. Cells were cultured to 70% confluencyand used in experiments at passages 5-8. Primary MPCs were generatedfrom skeletal muscle biopsies obtained from 12-week-old male Fischer 344rats (Envigo, Indianapolis, Ind.) as previously described⁷. In brief,fresh muscle tissue was placed in myogenic growth medium (MGM), whichconsisted of DMEM supplemented with 1% PSF-1, 20% fetal bovine serum,and 0.1% chick embryo extract (Accurate Chemicals, Westbury, N.Y.).Muscle was minced and digested in 0.2% collagenase type I (EMDMillipore, Temecula, Calif.) at 37° C. for 2 hours. Digested tissue wasfiltered through a 100 μm cell strainer and washed 3 times with MGM.Resulting muscle fibers were suspended in MGM, plated onto untreated 100mm petri dishes (Fisher Scientific), and cultured overnight at 37° C.within a humidified environment of 5% carbon dioxide in air. Thesupernatant was removed the next morning and transferred to cultureflasks (Corning Life Sciences, Corning, N.Y.). Resultant primary muscleprogenitor cells were cultured to 70% confluency and used in experimentsat passages 3 to 5.

Example 4: Conventional Casting of Tissue Constructs

Tissue constructs were prepared by conventional casting methodsresulting in composites comprising randomly organized cells andcollagen-fibril scaffolds. Rat primary MSCs prepared according toExample 3 were suspended at a density of 106 cells/mL in neutralizedoligomer solutions (1.5 mg/mL). Neutralization was achieved usingmulti-step or single-step procedures and reagents as described inExample 1. Neutralized MSC-oligomer suspensions were maintained at 4° C.prior to use. The MSC-oligomer suspension was aliquoted into a 24-wellplate (500 μL/well) and subsequently polymerized at 37° C. for 10minutes. Once polymerized, constructs were cultured for 24 hours inDulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin,streptomycin, amphotericin B (PSF-1; HyClone, Logan, Utah) and 10% fetalbovine serum (HyClone; Logan, Utah) at 37° C. within a humidifiedenvironment of 5% carbon dioxide in air. Tissue constructs were fixed in3% paraformaldehyde of culture and stained with phalloidin forvisualization of the actin cytoskeleton. For 3D qualitative analysis,tissue constructs were imaged using an Olympus FluoView FV-1000 confocalsystem adapted to an inverted microscope (IX81, Olympus Corporation,Tokyo, Japan). Confocal image stacks were processed using Imarissoftware and images analyzed on ImageJ for alignment using theDirectionality algorithm. As shown in FIG. 3D-F, tissue constructsformed by conventional casting methods exhibited randomly organizedcells and collagen fibril scaffolds.

Example 5: Preparation of Aligned Muscle Constructs by Extrusion

Acidic oligomer solutions were prepared and standardized as described inExample 1. Neutralized oligomer solutions were prepared according toExample 2 at a final concentration of 1.5 mg/mL and maintained at 4° C.to slow polymerization rate. MPCs were then suspended in the neutralizedoligomer solution at 10⁷ cells/mL and maintained at 4° C. prior to use.The 4° C. temperature was maintained by placing the solutions andsuspensions on ice. The MPC-oligomer suspension (500 μL) was drawn upinto a syringe and then extruded (FIG. 1) from the syringe (3 cc) at arate of 2 mL/min into an 4-mm diameter cylinder mold prepared from Ultem(FIG. 2A and open on both ends), which was maintained at 37° C. andstayed in the mold for about 10 minutes to form “constructs”.Afterwards, the constructs were transferred to a customized culturechamber and secured in tension with fasteners (FIG. 4A) and cultured inDMEM supplemented with 10% fetal bovine serum and 1% PSF-1 at 37° C. in5% carbon dioxide in air. After 24 hours, constructs were stained withphalloidin (actin) and Draq5 (nuclei) and analyzed by confocalmicroscopy to visualize solid collagen-fibril scaffold and cells withinthe constructs. Confocal image stacks were analyzed on ImageJ foralignment using the Directionality algorithm. As indicated in FIG. 3,solid collagen fibrils (FIG. 3A) and cells (FIG. 3B) were highly alignedparallel (FIG. 3C) to the direction of flow. By contrast, constructsprepared by conventional casting methods (Example 4) exhibited randomorganization (FIG. 3F) of solid collagen fibrils (FIG. 3D) and cells(FIG. 3E).

Example 6: Effect of Cell Density and Oligomer Concentration onEngineered Muscle Constructs Prepared by Extrusion

The same extrusion process described in Example 5 was applied for C2C12myoblasts at densities of 10⁶ and 10⁷ cells/mL as set forth in thisExample 6. C2C12 myoblasts were suspended at each density in neutralizedoligomer solutions (Example 1) prepared at either 1.3 mg/mL or 3.3mg/mL. Following extrusion, constructs were cultured within the customculture device for 14 days under passive tension (FIG. 4A) prior tofixation and staining with phalloidin and Draq5 for visualization ofcellular actin and nuclei, respectively. As indicated in FIG. 4,constructs prepared at high cell density and low oligomer concentration(Panel c of FIG. 4B) exhibited the necessary cell-cell interactions andcollagen-fibril scaffold support to facilitate a high level of cellfusion, nuclear alignment, and myotube formation. By contrast,constructs prepared at low cell density and high oligomer concentration(Panel b of FIG. 4B) exhibited the least amount of cell fusion andalignment largely owing to the increased solution viscosity and densityof solid fibrils formed during polymerization. Follow-up verificationstudies revealed that this fabrication strategy was also applicable toprimary rat MPCs, yielding highly reproducible engineered muscle withviable cells distributed throughout the construct after 2 weeks ofculture (FIG. 5).

Example 7: Generation Of Aligned Muscle Construct with Motor EndplateExpressing MPCs (MEE)

The same extrusion process outlined in Example 6 was applied to createaligned muscle constructs where the MPCs were induced to express motorendplates. Briefly, following extrusion constructs were cultured for 5days after which time motor endplate expression was induced by addingacetylcholine chloride (40 nM; Tocris Bioscience, Bristol, England),agrin (10 nM; R&D Systems, Minneapolis, Minn.), and neuregulin (2 nM;R&D Systems) to the culture medium. Constructs were cultured anadditional 7 days with medium changes every 3 days. Motor endplateexpression was confirmed by immunostaining with Alexa Fluor 594conjugated α-bungarotoxin (Molecular Probes, Eugene, Oreg.).

Example 8: Aligned Tissue Constructs Prepared by Extrusion of HumanAdipose-Derived Stem Cells (ASCs) Suspended in Polymerizable OligomerSolutions

The same extrusion process described in Example 5 was applied to humanadipose-derived stem cells (ASCs). ASCs represent an attractiveautologous cell type for tissue engineering and regenerative medicineapplications since they can be easily harvested from subcutaneous fatvia conventional liposuction techniques. ASCs represent a mesenchymalstem cell source with self-renewal property and multipotentialdifferentiation. Said differently, these cells are exhibit a highproliferative capacity and the ability to differentiate into multipledifferent cell types, including adipocytes, osteoblasts, tenocytes,myocytes, and neurocytes. Low passage human ASCs (Lonza) were culturedwithin collagen oligomer-coated flasks and maintained in complete mediumcomprising EGM-2 supplemented with 12% Hyclone FBS. Prior to extrusion,ASCs were suspended at a density of 500,000 cell/mL in neutralizedoligomer solution (1.5 mg/mL). For comparison purposes, randomlyorganized oligomer-ASC constructs were prepared within standard 96-wellplate using conventional casting methods as described in Example 4.Following culture for 24 hours, constructs were fixed inparaformaldehyde, stained with phalloidin (F-actin) followed by Draq5(nuclear) for 30 minutes at room temperature, and imaged using confocalmicroscopy. Confocal image stacks were processed using Imaris softwareand images analyzed on ImageJ for alignment using the Directionalityalgorithm. As shown in FIG. 6A-B, tissue constructs prepared byextrusion showed uniform alignment of cells in parallel with thedirection of flow. By contrast, tissue constructs prepared byconventional casting techniques exhibited randomly organized cells withno preferred directionality (FIG. 6C-D). Such randomly organized cellsfail to recapitulate the structure and functional properties of nativeskeletal muscle.

Example 9: Creation of Aligned Collagen-Fibril Construct Via Extrusion

The same extrusion process described in Example 5 was applied tooligomer solutions in absence of cells as set forth in this Example 9.Stock oligomer solution was diluted with 0.01N hydrochloric acid andneutralized as described in Example 1 to achieve a neutralized oligomersolution at a concentration of 3.3 mg/ml. The neutralized oligomersolution was kept on ice (4° C.) prior to induction of polymerization.Oligomer solution (500 μL) was extruded from a syringe (3 cc) at a rateof 2.1 mL/min into an Ultem 4-mm diameter cylinder mold, which wasmaintained at 37° C. Following extrusion, the construct was imaged viaconfocal reflection microscopy for visualization of the solid fibrilarchitecture. As shown in FIG. 7, the resultant fibrillar collagenscaffold was highly aligned.

Example 10: Laryngectomy and Implantation of Engineered Muscle

Engineered muscle constructs formed by extrusion of primary rat MPCssuspended within neutralized oligomer solutions were evaluated in anestablished rat partial laryngectomy model. Neutralized oligomersolutions were prepared as described in Example 1 at a finalconcentration of 1.3 mg/mL. Primary rat MPCs were generated according toExample 3 and suspended in the neutralized oligomer solution at adensity of 10E7 cells/mL. The suspension was extruded to formMPC-oligomer constructs as described in Example 6. Constructs werecultured for 5 days with medium changes every 2 days. On day 5, mediumwas changed to differentiation medium, representing DMEM supplementedwith 8% horse serum (HyClone) and 1% PSF-1. Constructs were cultured foran additional 7 days to induce myotube formation. MEE-oligomerconstructs were extruded as described above and cultured as described inExample 7. Oligomer only constructs were prepared by extrusion ofneutralized oligomer solutions (1.3 mg/mL) in absence of cells.

The animal study protocol was approved by Purdue Animal Care and UseCommittee, and institutional guidelines, in accordance with the NationalInstitutes of Health guidelines, were followed for the handling and careof the animals. 12 Fisher 344 rats were anesthetized withintraperitoneal injection of xylazine and ketamine and then maintainedon 1-4% isoflurane. The ventral larynx was exposed via a midlineincision. The sternohyoid muscle was incised and reflected to expose thethyroid cartilage. A section (approximately 2 mm×2 mm) of thyroidcartilage and associated adductor muscle was removed from the left side.Animals were randomized into the following experimental groups:MPC-oligomer construct (n=4), MEE-oligomer construct (n=4), oligomerconstruct only (n=2), and defect only control (n=2). The sternohyoidmuscle was reapposed and sutured, and subcutaneous tissue and skinclosed with 5-0 Vicryl suture.

Example 11: Laryngectomy in the Presence and Absence of RecurrentLaryngeal Nerve Injury and Implantation of Engineered Muscle andCartilage

Engineered cartilage and muscle constructs were implanted in anestablished rat partial laryngectomy model in the presence and absenceof recurrent laryngeal nerve injury. Engineered muscle constructs wereformed by extrusion of primary rat MPCs suspended within neutralizedoligomer solutions. Neutralized oligomer solutions were prepared asdescribed in Example 1 at a final concentration of 1.3 mg/mL. Primaryrat MPCs were generated according to Example 3 and suspended in theneutralized oligomer solution at a density of 10E7 cells/mL. Thesuspension was extruded to form MPC-oligomer constructs as described inExample 6. Constructs were cultured for 5 days with medium changes every2 days. On day 5, medium was changed to differentiation medium,representing DMEM supplemented with 8% horse serum (HyClone) and 1%PSF-1. Constructs were cultured for an additional 7 days to inducemyotube formation. MEE-oligomer constructs were extruded as describedabove and cultured as described in Example 7.

The animal study protocol was approved by Purdue Animal Care and UseCommittee, and institutional guidelines, in accordance with the NationalInstitutes of Health, were followed for the handling and care of theanimals. Fisher 344 rats were anesthetized with intraperitonealinjection of xylazine and ketamine and then maintained on 1-4%isoflurane. The ventral larynx was exposed via a midline incision. Thesternohyoid muscle was incised and reflected to expose the thyroidcartilage. A section (approximately 2 mm×2 mm) of thyroid cartilage andassociated adductor muscle was removed from the left side. Animals wererandomized into groups receiving engineered constructs with MPC or MEEcells with and without RLN injury. All groups received identicalengineered cartilage implants with endpoints at 1, 3, and 6 months. Themuscle construct was placed into the defect (similar to a medializationlaryngoplasty implant), followed by the cartilage construct over topwith extrusion prevented by suturing overlying sternohyoid muscles overthe cartilaginous defect. For groups with RLN injury, the left recurrentlaryngeal nerve was cauterized as it entered the larynx. Thesubcutaneous tissue and skin were then closed with 5-0 Vicryl suture.

Example 12: Video Laryngoscopy and Laryngeal Electomyography (EMG)

Video laryngoscopy and/or laryngeal electromyography were performed onanesthetized rats at 1, 3, and 6 month timepoints following partiallaryngectomy and reconstruction as described in Examples 10 and 11.Video laryngoscopy was performed using a rigid endoscope with attachedcamera. Electromyogram (Niking Viking Quest electromyography machine,Madison, Wis.) was used with a 25-gauge bipolar concentric needle,settings with amplitude of 50 to 100 μV, 10- to 100-ms sweep speeds, anda grounding clamp at the exposed lateral sternocleidomastoid muscle. TheEMG recording needle was inserted directly into the center of thedefect/implant site, the adductor (TA) muscle complex, and the posteriorcricoarytenoid (PCA) muscle during laryngospasm and at rest. Immediatelyfollowing laryngoscopy and electromyography procedures, rats werehumanely euthanized and tissue collected.

Example 13: Histopathological and Histochemistry Assessment

Histopathological analysis was performed at 1, 3, or 6 month timepointsfollowing partial laryngectomy and reconstruction as described inExamples 10 and 11. After euthanasia, rat larynges and associatedimplants were harvested en bloc, fixed in 4% paraformaldehyde overnight,and then transferred to 30% sucrose at 4° C. for an additional 24 hours.Cryosections (25 μm thickness) were prepared on a Thermo Cryotome FE(Fisher Scientific, Kalamazoo, Mich.). Sections were stained withhematoxylin & eosin (H&E) and Alcian blue for histopathologicalanalysis. Slides were viewed on a Nikon upright microscope (EclipseE200, Nikon, Melville, N.Y.) and images captured with a Leica camera(DFC480, Leica Buffalo Groove, Ill.). Myofiber diameter was measuredusing Image J software (NIH).

For histochemistry analysis, all specimens were washed with phosphatebuffered saline (PBS) 3 times, permeabilized with 0.1% Triton X-100 for20 minutes, and blocked with 1% BSA for 2 hours. Whole engineered muscleconstructs were incubated overnight at 4° C. with Alexaflour 488conjugated phalloidin (1:25 Molecular Probes, Eugene, Oreg.) forvisualization of F-actin and counterstained with Draq5 (1:1000 CellSignaling, Danvers, Mass. 4084L) nuclear stain. For staining tissueexplant cryosections, beta III tubulin conjugated primary antibody (1:10NL647 Molecular Probes) was applied and incubated overnight at 4° C.After rinsing extensively, slides were incubated with Alexa Fluor 594conjugated α-bungarotoxin (1:100) for 2 hours at room temperature.Slides were rinsed and mounted with Fluorogel for imaging on an OlympusFluoview confocal microscope (IX81, Olympus Waltham, Mass.) or Zeiss LSM880 confocal microscope (Oberkochen, Germany).

Example 14: Reconstruction of Laryngeal Defects with MPC-OligomerConstructs Yields Myogenesis, Neurovascular Regeneration, and TissueIntegration in Absence of an Inflammatory-Mediated Foreign Body Response

Laryngectomy and implantation of engineered muscle was performed asdescribed in Example 10. During the post-surgical period, all animalssteadily gained weight and showed no signs of laryngeal compromise. Asexpected, partial laryngectomy with resection of cartilage and muscleand no treatment resulted in a healing response marked by inflammationand fibrous tissue formation (scar tissue) within the defect area (FIG.8B). In contrast, oligomer implants, with and without MPCs, integratedrapidly with adjacent host tissues, showing no significant inflammatoryreaction or proteolytic degradation (FIG. 8B-E). A surprising findingwas that chondrogenesis accompanied muscle regeneration over time (FIG.8E). Compared to the oligomer only group, MPC-oligomer and MEE-oligomerconstructs showed more rapid muscle regeneration and maturation asevidenced by obvious striations within the implants as early as 1 monththat became more evident at 3 months. By 3 months, the relative extentof striated muscle increased, and was supported by neurogenesis, asevidenced by prominent beta III tubulin staining arising from thegraft-host tissue interface (FIG. 10). Interestingly, results based onbeta III tubulin and α-bungarotoxin staining indicated that the greatestlevel of innervation was achieved with the MEE-oligomer group.Qualitative EMG measurements provided further corroborating evidence ofenhanced innervation of the MEE-oligomer constructs, with theMEE-oligomer group demonstrating the greatest level of motor unitactivity (recruitment) during laryngospasm (FIG. 9).

Results of these studies suggest that aligned MEE-oligomer constructscontributed to rapid tissue integration and regeneration in the absenceof any significant inflammatory reaction or rapid proteolyticdegradation, yielding restored muscle with enhanced innervation onhistology, and functional elicitation of motor unit potentials. Majoradvantages of the model include the use of autologous cells, alignedmuscle constructs for rapid restoration of vascularization, innervation,and function, and a scalable fabrication method that can be translatedinto patient-specific designs.

Example 15: Reconstruction of Denervated Hemilarynx after PartialLaryngectomy with Engineered Cartilage and Muscle Constructs YieldsMyogenesis, Neurovascular Regeneration, and Functional TissueRestoration in Absence of an Inflammatory-Mediated Foreign Body Response

Laryngectomy in the presence and absence of recurrent laryngeal nerveinjury and implantation of engineered muscle and cartilage as describedin Example 11. All animals survived the post-surgical period with nolife-threatening complications. Some RLN injured animals showed mildstridor but this resolved with time. All animals steadily gained weightover the study period. Post-mortem gross pathological exam showedintegration of the cartilage and muscle implants into host tissue withno macroscopic signs of inflammation. Alcian blue staining ofcryo-sectioned specimens was weakly positive at 1 month (FIG. 11B). At 3and 6 month timepoints, sections demonstrated progressive increase inAlcian blue staining consistent with glycosaminoglycan deposition andcartilage formation (FIG. 11C-D). H&E staining at 1 month displayedimmature muscle neighboring native adductor muscle with no foreign bodyresponse (FIG. 12A,D). At 3 months, maturation of the muscle within theimplant was demonstrated by development of cross striations and myofiberalignment (FIG. 12B,E). By 6 months, little to no immature muscle wasvisible within the defect area suggesting complete integration withnature tissue (FIG. 12C-F).

α-bungarotoxin and beta III tubulin staining demonstrated that implantsthat were induced to form motor endplates (MEE) showed a great number ofmotor endplates and robust innervation (FIG. 13). Qualitative EMGmeasurements of the adductor muscle complex of the MPC-oligomer treatedanimals showed low level activity (FIG. 14A,B) whereas the MEE-oligomertreated animals showed near normal muscle activity, with interferenceand recruitment patterns (during laryngospasm) that mimicked normaladductor muscle (FIG. 14C,D). This finding was further corroborated byadductor muscle complex myofiber diameter measurements, with values forMEE-oligomer treated animals significantly greater than those forMPC-oligomer treated animals (p<0.001 FIG. 14E).

In nerve injured animals, video laryngoscopy showed function recovery in100% of animals receiving the MEE implant with definitive althoughslightly delayed movement at all timepoints. By contrast, none of theanimals receiving the MPC implant showed definitive movement at anytimepoint (FIG. 15).

REFERENCES

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We claim:
 1. A solid collagen construct comprising aligned collagenfibrils and aligned cells.
 2. A solid collagen construct prepared by theprocess of extruding a polymerizable collagen solution with an extruderto generate the solid collagen construct.
 3. The solid collagenconstruct of claim 2, wherein the polymerizable collagen solution is apolymerizable collagen oligomer solution.
 4. The solid collagenconstruct of claim 3, wherein the solution is extruded into a container.5. The solid collagen of claim 4, wherein the container is at a highertemperature than the extruder.
 6. The solid collagen construct of claim5, wherein the extruder is at about 4° C. and the container is at about37° C.
 7. The solid collagen construct of claims 2-6, wherein theextruder is a syringe.
 8. The solid collagen construct of claims 3-7,wherein the polymerizable collagen oligomer solution comprises collagenoligomer, water, an acid, one or more salts, and a base.
 9. The solidcollagen construct of claim 8, wherein the polymerizable collagenoligomer solution further comprises a sugar.
 10. The solid collagenconstruct of claims 8-9, wherein the acid is HCl and the base is NaOH.11. The solid collagen construct of claims 8-10, wherein the one or moresalts are KH₂PO₄, Na₂HPO₄, KCl, and NaCl.
 12. The solid collagenconstruct of claims 9-11, wherein the sugar is glucose.
 13. The solidcollagen construct of claims 2-11, wherein the concentration of thecollagen in the polymerizable collagen solution is between about 0.1mg/ml and about 40 mg/ml.
 14. The solid collagen construct of claim 13,wherein the concentration of the collagen in the polymerizable collagensolution is between about 1 mg/ml and about 10 mg/ml.
 15. The solidcollagen construct of claim 14, wherein the concentration of thecollagen in the polymerizable collagen solution is between about 2 mg/mland about 6 mg/ml.
 16. The solid collagen construct of claim 15, whereinthe concentration of the collagen in the polymerizable collagen solutionis between about 3 mg/ml and about 5 mg/ml.
 17. The solid collagenconstruct of claims 2-16, wherein the polymerizable collagen solution isextruded at a rate of between about 1 ml/minute and about 3 ml/minute.18. The solid collagen construct of claim 16, wherein the polymerizablecollagen solution is extruded at a rate of about 2 ml/minute.
 19. Thesolid collagen construct of claims 2-18, wherein the pH of thepolymerizable collagen solution is between about 4 and about
 10. 20. Thesolid collagen construct of claim 19, wherein the pH of thepolymerizable collagen solution is between about 6 and
 8. 21. The solidcollagen construct of claim 20, wherein the pH of the polymerizablecollagen solution is about 7.4.
 22. The solid collagen construct ofclaims 2-21, wherein the solid collagen is fibrillar.
 23. The solidcollagen construct of claims 2-22, wherein the polymerizable collagensolution polymerizes during extrusion in the extruder.
 24. The solidcollagen construct of claims 2-23, wherein the polymerizable collagensolution polymerizes after extrusion from the extruder.
 25. The solidcollagen construct of claims 4-24 wherein the container is a die ormold.
 26. The solid collagen construct of claim 26, wherein thecontainer is a mold.
 27. A solid collagen construct prepared by theprocess comprising extruding a suspension of polymerizable collagensolution and cells with an extruder to generate a solid collagenconstruct wherein the solid collagen construct is embedded with cells.28. The solid collagen construct of claim 27, wherein the polymerizablecollagen solution is a polymerizable collagen oligomer solution.
 29. Thesolid collagen construct of claims 27-28, wherein the cells are MPCcells.
 30. The solid collagen construct of claims 27-28, wherein thecells are MEE cells.
 31. The solid collagen construct of claims 27-28,wherein the cells are ASC.
 32. The solid collagen construct of claims27-31, wherein the polymerizable collagen oligomer suspension isextruded into a container.
 33. The solid collagen construct of claim 32,wherein the container is at a higher temperature than the extruder. 34.The solid collagen construct of claim 33, wherein the extruder is atabout 4° C. and the container is at about 37° C.
 35. The solid collagenconstruct of claims 27-34, wherein the extruder is a syringe.
 36. Thesolid collagen construct of claims 28-35, wherein the polymerizableoligomer solution comprises collagen oligomer, water, an acid, one ormore salts, and a base.
 37. The solid collagen construct of claim 36,wherein the polymerizable collagen oligomer solution further comprises asugar.
 38. The solid collagen construct of claims 36-37, wherein theacid is HCl and the base is NaOH.
 39. The solid collagen construct ofclaims 36-38, wherein the one or more salts are KH₂PO₄, Na₂HPO₄, KCl,and NaCl.
 40. The solid collagen construct of claims 37-39, wherein thesugar is glucose.
 41. The solid collagen construct of claims 27-40,wherein the concentration of the collagen in the polymerizable collagensolution is between about 0.1 mg/ml and about 40 mg/ml.
 42. The solidcollagen construct of claim 41, wherein the concentration of thecollagen in the polymerizable collagen solution is between about 1 mg/mland about 6 mg/ml.
 43. The solid collagen construct of claim 42, whereinthe concentration of the collagen in the polymerizable collagen solutionis between about 3 mg/ml and about 5 mg/ml.
 44. The solid collagenconstruct of claims 27-43, wherein the polymerizable collagen suspensionis extruded at a rate of between about 1 ml/minute and about 3ml/minute.
 45. The solid collagen construct of claim 44, wherein thepolymerizable collagen suspension is extruded at a rate of about 2ml/minute.
 46. The solid collagen construct of claims 27-45, wherein thepH of the polymerizable collagen solution is between about 4 and about10.
 47. The solid collagen construct of claim 46, wherein the pH of thepolymerizable collagen solution is between about 6 and
 8. 48. The solidcollagen construct of claim 47, wherein the pH of the polymerizablecollagen solution is about 7.4.
 49. The solid collagen construct ofclaims 27-48, wherein the solid collagen is fibrillar.
 50. The solidcollagen construct of claims 27-49, wherein the polymerizable collagensuspension polymerizes during extrusion in the extruder.
 51. The solidcollagen construct of claims 27-50, wherein the polymerizable collagensuspension polymerizes after extrusion from the extruder.
 52. The solidcollagen construct of claims 32-51 wherein the container is a die ormold.
 53. The solid collagen construct of claim 52, wherein thecontainer is a mold.
 54. The solid collagen construct of claims 27-53further comprising the step of culturing the solid collagen construct.55. The solid collagen construct of claims 27-54, wherein the solidcollagen construct is in the form of tissues with aligned architectures.56. The solid collagen construct of claim 55, where the tissue isselected from cardiac muscle, nerve, smooth muscle, tendon, andligament.
 57. The solid collagen construct of claim 56, wherein thetissue is a muscle.
 58. The solid collagen construct of claim 57,wherein the muscle is skeletal muscle, adductor muscle, cardiac muscle,or smooth muscle.
 59. Tissue implants for human or veterinary usecomprising the solid collagen constructs of claims 2-58.
 60. Tissueimplants of claim 59 wherein the tissue is selected from cardiac muscle,nerve, smooth muscle, tendon, and ligament.
 61. The tissue implants ofclaim 60, wherein the tissue is a muscle.
 62. The tissue implants ofclaim 61, wherein the muscle is skeletal muscle, cardiac muscle, orsmooth muscle.
 63. A process of preparing solid collagen constructscomprising extruding a polymerizable collagen solution with an extruderto generate a solid collagen construct.
 64. The process of claim 3,wherein the polymerizable collagen solution is a polymerizable collagenoligomer solution.
 65. The process of claim 64, wherein the solution isextruded into a container.
 66. The solid collagen of claim 65, whereinthe container is at a higher temperature than the extruder.
 67. Theprocess of claim 66, wherein the extruder is at about 4° C. and thecontainer is at about 37° C.
 68. The process of claims 63-67, whereinthe extruder is a syringe.
 69. The process of claims 64-68, wherein thepolymerizable collagen oligomer solution comprises collagen oligomer,water, an acid, one or more salts, and a base.
 70. The process of claim69, wherein the polymerizable collagen oligomer solution furthercomprises a sugar.
 71. The process of claims 69-70, wherein the acid isHCl and the base is NaOH.
 72. The process of claims 69-71, wherein theone or more salts are KH₂PO₄, Na₂HPO₄, KCl, and NaCl.
 73. The process ofclaims 70-71, wherein the sugar is glucose.
 74. The process of claims63-71, wherein the concentration of the collagen in the polymerizablecollagen solution is between about 0.1 mg/ml and about 40 mg/ml.
 75. Theprocess of claim 74, wherein the concentration of the collagen in thepolymerizable collagen solution is between about 1 mg/ml and about 10mg/ml.
 76. The process of claim 75, wherein the concentration of thecollagen in the polymerizable collagen solution is between about 2 mg/mland about 6 mg/ml.
 77. The process of claim 76, wherein theconcentration of the collagen in the polymerizable collagen solution isbetween about 3 mg/ml and about 5 mg/ml.
 78. The process of claims63-77, wherein the polymerizable collagen solution is extruded at a rateof between about 1 ml/minute and about 3 ml/minute.
 79. The process ofclaim 77, wherein the polymerizable collagen solution is extruded at arate of about 2 ml/minute.
 80. The process of claims 63-79, wherein thepH of the polymerizable collagen solution is between about 4 and about10.
 81. The process of claim 80, wherein the pH of the polymerizablecollagen solution is between about 6 and
 8. 82. The process of claim 81,wherein the pH of the polymerizable collagen solution is about 7.4. 83.The process of claims 63-82, wherein the solid collagen is fibrillar.84. The process of claims 63-83, wherein the polymerizable collagensolution polymerizes during extrusion in the extruder.
 85. The processof claims 63-84, wherein the polymerizable collagen solution polymerizesafter extrusion from the extruder.
 86. The process of claims 65-85wherein the container is a die or mold.
 87. The process composition ofclaim 86, wherein the container is a mold.
 88. A process for preparing asolid collagen construct comprising extruding a suspension ofpolymerizable collagen solution and cells with an extruder to generate asolid collagen construct wherein the solid collagen construct isembedded with cells.
 89. The process of claim 88, wherein thepolymerizable collagen solution is a polymerizable collagen oligomersolution.
 90. The process of claims 88-89, wherein the cells are MPCcells.
 91. The process of claims 88-89, wherein the cells are MEE cells.92. The process of claims 88-89, wherein the cells are ASC.
 93. Theprocess of claims 88-92, wherein the polymerizable collagen oligomersuspension is extruded into a container.
 94. The process of claim 93,wherein the container is at a higher temperature than the extruder. 95.The process of claim 94, wherein the extruder is at about 4° C. and thecontainer is at about 37° C.
 96. The process of claims 88-95, whereinthe extruder is a syringe.
 97. The process of claims 89-96, wherein thepolymerizable oligomer solution comprises collagen oligomer, water, anacid, one or more salts, and a base.
 98. The process of claim 97,wherein the polymerizable collagen oligomer solution further comprises asugar.
 99. The process of claims 97-98, wherein the acid is HCl and thebase is NaOH.
 100. The process of claims 97-99, wherein the one or moresalts are KH₂PO₄, Na₂HPO₄, KCl, and NaCl.
 101. The process of claims98-100, wherein the sugar is glucose.
 102. The process of claims 88-101,wherein the concentration of the collagen in the polymerizable collagensolution is between about 1 mg/ml and about 10 mg/ml and the solidcollagen construct forms fibrils.
 103. The process of claim 102, whereinthe concentration of the collagen in the polymerizable collagen solutionis between about 2 mg/ml and about 6 mg/ml.
 104. The process of claim103, wherein the concentration of the collagen in the polymerizablecollagen solution is between about 3 mg/ml and about 5 mg/ml.
 105. Theprocess of claims 88-104, wherein the polymerizable collagen suspensionis extruded at a rate of between about 1 ml/minute and about 3ml/minute.
 106. The process of claim 105, wherein the polymerizablecollagen suspension is extruded at a rate of about 2 ml/minute.
 107. Theprocess of claims 88-106, wherein the pH of the polymerizable collagensolution is between about 4 and about
 10. 108. The process of claim 107,wherein the pH of the polymerizable collagen solution is between about 6and
 8. 109. The process of claim 108, wherein the pH of thepolymerizable collagen solution is about 7.4.
 110. The process of claims88-109, wherein the solid collagen is fibrillar.
 111. The process ofclaims 88-110, wherein the polymerizable collagen suspension polymerizesduring extrusion in the extruder.
 112. The process of claims 88-111,wherein the polymerizable collagen suspension polymerizes afterextrusion from the extruder.
 113. The solid collagen composition ofclaims 93-112 wherein the container is a die or mold.
 114. The solidcollagen composition of claim 113, wherein the container is a mold. 115.The process of claims 88-114, further comprising the step of culturingthe solid collagen construct.
 116. The process of claims 88-115, whereinthe solid collagen construct is in the form of tissues with alignedarchitectures.
 117. The process of claim 116, where the tissue isselected from cardiac muscle, nerve, smooth muscle, tendon, andligament.
 118. The process of claim 117, wherein the tissue is a muscle.119. The process of claim 118, wherein the muscle is skeletal muscle,cardiac muscle, or smooth muscle.
 120. The solid collagen compositionsof claim 41, wherein the concentration of collagen in the polymerizablecollagen solution is between about 1 mg/ml and about 10 mg/ml.
 121. Thesolid collagen composition of claims 24 and 51, wherein polymerizationoccurs after extrusion from the extruder.
 122. The process of claims 85and 112 wherein polymerization occurs after extrusion from the extruder.123. A kit for tissue reconstruction comprising a polymerizable collagensolution or a polymerizable collagen suspension and a syringe.
 124. Thekit of claim 123, wherein the polymerizable collagen solution is asolution of polymerizable collagen oligomers.
 125. A kit for tissuereconstruction comprising a solid collagen construct and a mold.